Hiroshima Journal of Medical Sciences Vol. 30, No. 4, December, 1981

WJM 30-39

EFFECT OF BOVINE SERUM ALBUMIN ON THE ABSORPTION OF SULFONAMIDES IN THE RAT*>

By

Yutaka HIGASHI and Noboru YATA

Institute of Pharmaceutical Sciences, Hiroshima University School of Medici11e

(Received September 2, 1981)

ABSTRACT

Effect of bovine serum albumin (BSA} on the absorption of sulfonamides was studied employing in situ recirculating perfusion method in the rat. The absorption rate constant of sulfamethoxazole was greatly decreased by addition of BSA to the perfu­sion solution. But, the decreasing effect of BSA on the absorption rate was diminished after removing BSA from perfusion solution. The decreasing effect of BSA on the absorption rate constant of sulfanilamide was not observed. The binding of sulfonamides to BSA was studied using an equilibrium dialysis method. Sulfamethoxazole was bound to large extent, whereas sulfanilamide was little bound. The bound fraction of sulfamethoxazole increased as the concentration of BSA increased. Influence of BSA on permeability of intestinal mucosa was not observed. Thus, the decreasing effcet of BSA on the absorption rate constant of sulfamethoxazole was attributed mainly to the binding to BSA which results in the decrease of unbound drug permeable to mucosal membrane. However, experimental results were slightly deviated from the theoretical value calculated by assuming that bound drug was unabsorbed from the intestinal mucosa.

287

INTRODUCTION

An orally administered drug must be absorbed from gastrointestinal tract to an extent and at a rate that will result in circulating drug levels sufficient to elicit a pharmacological response of desired magnitude and duration. The efficien­cy with which a drug is absorbed is a function of many variables1>. Furthermore, absorption of drugs have been reported to be affected by another drugs, pharmaceutical additives or food administered concomitantly2-rn. The in­teraction of drug with another drug, with non­medicinal component of dosage form or with

substances normally found in biological systems (e.g., in the gastrointestinal tract) can cause the formation of complexess-rn, resulting in the increase or decrease of drug absorption. Such complexes may be essentially unabsorb­able if they are practically lipoid-insoluble and large in size. In the present study, effect of complexation to macromolecule on the absorp­tion of drugs were investigated. As models of drug and macromolecule, sulfonamides and bo­vine serum albumin (BSA) were used. The absorption of drug from aqueous solution with or without BSA was studied employing in situ recirculating perfusion method in the rat.

288 Y. Higashi et al.

EXPERIMENTAL

Materials--Commercially available sulfa­nilamide and sulfatnethoxazole were U!Sed with­out further purification. Bovine serum albumin (Fraction V) (BSA) was purchased from· the · Armour Pharmaceutical Company and cello­phane tubing (Visking Company, 27 /32 inflated diameter) was used as a dialyzing membrane. All other chemicals were analytical grade.

Procedure of In situ Absorption Experiment --Male Sprague-Dawley rats weighing 200 to 300 g were fasted for about 20 hrs prior to the experiments but water was allowed ad libitum. The animals were anesthetized with pentobarbital and the small intestine was ex­posed by a midline abdominal incision. The stomach and cecum were ligated by cannula~

tion below the duodenum and at the upper portion of ileocecurn with glass cannulae con­nected to polyethylene tube. The intestine was replaced in the abdomen, and incision closed. The small intestine was first . cleared of partic­ulate matter with 100 ml of drug-free saline solution .previously warmed to 37° C. · · The per­fusion solution was perfused with a cum pump (Tokyo Rika Kikai, C-16) at a rate of 5 ml/ min, and was maintained at 37° C. Phosphate­citrate isotonic buffer, pH 6. 5 was used as a perfusion solution. ·

To determine the absorption rate constant of sulfonamides, the following perfusion procedure was used. ·sixty ml of perfosiOn solution con­taining drug and phenol red as a volume indi­cator was perfused. The initial 20 ml of per­fusion solution after perfusion was discarded and remaining · 40 ml was . recirculated. . Five min after the . start of recirculation; 0. 5 ml of perfusion solution was withdrawn from reservor as a 0 time sample, and then, at designated time, 0. 5 ml of sample was withdrawn for 30 min. Sulfona.mides and phenol red . were anal­ysed . separately. Unabsorbed .fraction in per­fusion solution at time t, D t, was calculated with equation 1.

Dt=_!;_x_&_ (Eq. l) · Co It

w.here, C0 and C 1 are drug concentrations in · the perfusion solution at time 0 and t, respec-. tively, Io and I, are concentrations of phenol red used as a volume indicator, at time 0 and t, respectively. Logarithmic plot of unabsorbed

fraction against time resulted in a straight line. This finding shows that the absorption obey the pseudo first-order kinetics. Then, the fol­lowing equation will be. derived.

log Dt =log Do- kaPP •t . 2.303

(Eq. 2)

Apparent pseudo first-order absorption rate con­stant, kaPP' was calculated from the slope of the line which was obtained using least squares analysis.

Binding to Bovine Serum Albumin--BSA was dissolved in a phosphate-citrate isotonic buffer (pH 6. 5) used in absorption experiments. The binding of drugs to BSA was determined with an equilibrium dialysis method employing the cellophane bag. Ten ml of the BSA solu­tion was pipetted in a cellophane bag. The bag . was placed in a glass tube containing 10 ml of the buffer solution of drug at various concentrations. After continuous horizontal shaking of 90 cycle/min for 8 hrs at 37°C in a water bath shaker, the concentration of drug outside the bag was measured. · It took 6 hrs to establish a dialysis equilibrium. In the pre­liminary experiment's, it was determined that no sulfonamides was bound to the cellophane tubing over the range of concentration studied.

Results were plotted according to the· method of Scatchard12>, using the relationship:

;1 =K·n-K~r (Eq. 3)

where, r is number of moles of drug bound per mole of protein, C 1 is molar concentration of unbound drug, K is association constant (litter/mole), and n is number of drug-binding sites per molecule of protein. The value of 69000 was used as the molecular weight of BSA13>. Bound fraction, fi, of sulfonamides in the perfusion solution (total (bound and unbound) ·concentration of 1 mM) is unable to be determined directly by the method of equi­librium dialysis, then was calculated using the n and K at a respective concentration of BSA, emplc:)ying equ.ation 4.

f1 =Cb c, n·P, +Ct +K---1 -{(n·P, +Ct +K-1 )2

-4n·Pt ·C ,}112 2·C 1

(Eq. 4) where, Cb and C, are bound and total concen­tration of drug, respectively, and P 1 is molar concentration of albumin.

Effect of BSA on . the Absorption of Sulfonamides 289

Analytical Methods--Sulfonamides were colorimetrically assayed employing a modified Bratton-Marshall method with Tsuda's rea­gent14l. Phenol red was analysed colorimetric­ally at 545 nm after alkalization with NaOH.

RESULTS

Effect of Bovine Serum Albumin on the Absorption of Sulfonamides through the Rat Small Intestine

Absorption of sulfonamides through rat small intestine was studied employing the in situ recirculating perfusion method at pH 6. 5. The fraction of unabsorbed sulfamethoxazole and sul­fanilamide in the recirculating . perfusion solution were plotted in logarithmic scale against time. Straight lines were obtained (not shown). Fur­thermore, concentration-dependent change of absorption rate constant of sulfamethoxazole was not observed at a concentration range of 0. 5 to 2. 0 mM (Table 1). These findings indi­cate that absorption processes of sulfonamide are performed through a passive diffusion, at the concentrations studied.

Table I. The Absorption Rate Constant of Sulfame­thoxazole in Rat Small Intestine

Concn. •> (mM)

0.5 1.0

2.0

12.21±0.98

12.05±0.45

13.09±0.78

5

10

5

a ) initial concentration · of sulfamethoxazole b) absorption rate constant c ) number of experiments

Effect of BSA on the absorption of sulfon­amides were studied at a drug concentration of 1 mM. Fig. 1 is a plot of absorption rate constant of sulfamethoxazole as a function of BSA concentration. A decrease in the absorp­tion rate constant was observed as the BSA concentration increased. To study the physio­logical change of intestinal mucosa, further ex­periments were performed. Perfusion solution containing sulfamethoxazole (1 mM) and BSA (1. 25 .w /v %) was perfused for 20 min and the absorption· rate constant was determined. And then, the perfusion solution was replaced with that containing drug without BSA, followed by the same perfusion procedure to determine the absorption rate constant of sulfamethoxazole

4

2

0 0 0.5 1.0 1.5 2.0 2.5 Concn. of BSA (w/v%)

Fig. 1. Concentration Effect of BSA on the Ahsorp-· tion Rate Constant of Sulfamethoxazole in the Rat at pH6.5 Initial concn. of sulfamethoxazole : 1 mM Each value represents the mean±S. E. fo1 four to. ten experiments. * p<(0.05 ** p<(0.01

0 10 20 10 20 Time, min

Fig. 2. Logarithmic Plot of Unabsorbed Fraction of Sulfamethoxazole with or without BSA in the Same Rat Arraw indicates the replacement of perfusion solution containing BSA to those without BSA.

-O-: with BSA (1.25w/v%) -0- : without BSA

without BSA. As shown in Fig. 2, which represents the typical plot, the decreased rate of absorption with BSA was recovered near to the value for control rat after replacement . with perfusion solution without BSA. With respect to the absorption rate constant of sul­fanilamide, the decreasing effect of BSA was not observed as shown in Fig. 3.

Binding of Sulfonamides to Bovine Serum Albumin

It is well known that su_l£onamides bind to

290 Y. Higashi et al.

12

2

0 0 ~5 LO L5 2.0 Concn. of BSA (w/v%)

Fig. 3. Concentration Effect of BSA on the Absorp­tion Rate Constant of Sulfanilamide in the Rat at pH 6.5 Initial concn. of sulfanilamide : 1 mM Each value represents the mean±S. E. for three to eight experiments.

Table II. Binding Parameters of Sulfamethoxazole at 37°C, pH6.5

Pt•> nb>

0.25 2.2 0.5 2.1 1.0 2.1 2.0 2.1 2.5 2.0

a ) concentration of BSA (w/v%) b ) bindind sites per mole BSA c ) association constant (litter/mole)

Kc>

4730 4610 4520 3400 3150

plama protein, particularly to albumin15>. Bind­ing of sulfonamides to BSA were investigated as a model for macromolecule-drug complex employing an equilibrium dialysis method. Number of binding sites, n, and association constant, K, were calculated from Scatchard's equation (Eq. 3). For various concentrations of BSA, calculated numbers of binding sites and association constants were listed in Table II. Number of drug-binding sites was not in­fluenced by the concentration of BSA, but as­sociation constant was slightly decreased as the increase in BSA concentration. The calculated bound fractions were plotted against the con­centration of BSA at a total sulfonamide con­centration of 1 mM (Fig. 4). Sulfamethoxazole was bound to large extent, whereas, sulfanil­amide was little bound in the concentration range of BSA studied.

0.5

ct). 0.4 er ·~ 0.3 ~ '"' ~ 0.2

-g ::i

~ 0.1

0.5

0---0-0-

1.0 1.5 2.0 2.5 Concn. of BSA (w/v%)

Fig. 4. Concentration Effect of BSA on Bound Frac­tion of Sulfonamides, {3, at 37°C, pH 6. 5

-•-: sulfamethoxazole -0- : sulfanilamide

Total concn. of sulfonamide is 1 mM.

DISCUSSION

The absorption rate constant of sulfameth­oxazole was greatly decreased by addition of BSA to the perfusion solution as shown in Fig. 1. But, the decreasing effect of BSA on the absorption rate was diminished after remov­ing BSA from perfusion solution (Fig. 2). The decreasing effect of BSA on the absorption rate constant of sulfanilamide was not observed (Fig. 3). These findings indicate that BSA does not cause a irreversible damage to the rat small intestine and does not influence the permeability of mucosal membrane. The absorption rate constant of sulfamethoxazole decreased as bind­ing of the drug to BSA increased. Thus, the decreased absorption rate constant will be re­sponsible for the binding of the drug to BSA, leading to the decrease in concentration of un­bound drug. Since bound sulfonamide was reported to possess no antimicrobial activity16>, bound sulfonamide is thought to be impermeable to biomembrane. Assuming that the bound drug is impermeable to mucosa! membrane and only unbound drug is absorbed by intestinal tract, equation 5 is derived.

dDt = -k0 ·D1 = -k0 ·f·D, (Eq. 5) dt

where, f is the unbound fraction and k0 is absorption rate constant without BSA. Then, apparent absorption rate constant, k. Pp, is ex­pressed as follows:

kaPP=J·ko=(l-/3)•ko (Eq. 6) Therefore, a linear correlation would be ex-

Effect of BSA on the Absorption of Sulfonamides 291

Unbound Fraction, f

Fig. 5. Relations between the Absorption Rate Cons­tant of Sulfamethoxazole, kapp, in the presence or Absence of BSA and Unbound Fraction of Sulfame­thoxazole, f - - - - : theoretical line Each value represents the mean±S. E. for four to ten experiments.

pected when kaPP is plotted against unbound fraction, f or (1- ,8). But, as shown in Fig. 5, there is a slight deviation from the theoretical line calculated by assuming that bound drug is unabsorbable. This deviation suggests that the binding of sulfamethoxazole to BSA might be slightly affected in the intestinal tract or on the intestinal mucosa! memlJt'ane. It is not cleared here, and further investigations are required.

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